PROJECT SUMMARY/ABSTRACT This is a competing renewal application whose overall objective is to define the mechanisms of interferon (IFN)- signaling in malignant cells. We have provided the first evidence for the existence of a novel pathway engaged by the Type I IFN receptor (IFNR), involving the ULK1 kinase. This ULK1 pathway operates in an autophagy- independent manner to regulate expression of IFN stimulated genes (ISGs) and is essential for the generation of the suppressive effects of IFN??on primary malignant hematopoietic precursors from patients with myeloproliferative neoplasms (MPNs). Remarkably, ULK1 forms complexes with unique partners that have distinct structures and functional capabilities, suggesting that coordinated operation of such complexes is required for optimal control of IFN-induced antineoplastic effects. Aim 1 will identify upstream IFNR-generated signals and mechanisms that control ULK1 activation. It includes studies to map the IFN-phosphorylation sites on ULK1 and identify direct upstream kinases that modify its function. Aim 2 will define ULK1 effector pathways that mediate IFN-responses. It includes experiments to determine the roles of novel effectors that we have identified, including ROCK1/2, PCM1 and CARD9. A combination of approaches will be used, including generation of selective knockouts by the CRISPR-Cas9 methodology and mutation of putative ULK1 phosphorylation sites in the structures of target proteins, followed by assessment of its effects on IFN signaling and antineoplastic responses. Aim 3 will define the role of ULK1 pathways in the generation of the antineoplastic effects of IFN? in MPNs. Jak2V617F-driven MPN mouse models will be established using mice with Ulk1-/-, Pcm1-/-, or Card9-/- backgrounds, and the ability of IFN? to induce antineoplastic responses in such mice will be examined. The therapeutic effects of a ROCK1/2 pharmacological inhibitor alone and in combination with IFN??in mediating anti-MPN responses in vivo will be also determined. In other studies, the activation of IFN- dependent ULK1 effector signals will be determined in primary malignant precursors from patients with different MPNs participating in IFN-clinical trials and correlated with molecular characteristics and clinical responses to IFN?-treatment. Altogether, this proposal introduces highly novel concepts in the IFN-field and the mechanisms of IFN-generated antitumor effects and may lead to the development of novel therapeutic strategies and approaches for the treatment of MPNs.